AVALIAÇÃO MULTIFUNCIONAL DO PEPTÍDEO SINTÉTICO BIOINSPIRADO NA TOXINA TITYUS SERRULATUS

Description

(001) The present invention relates to the construction of a peptide rationally designed by advanced bioinformatics methods, which has been named Ts-MAP1, possessing direct activity against different pathogenic microorganisms, namely, bacteria: Acinetobacter baumannii (003324845 HRAN - Hospital Regional Asa Norte), Escherichia coli clinical isolate (3323742), Escherichia coli (KPC + 001812446), Klebsiella pneumoniae (ATCC + 13883), Klebsiella pneumoniae (KPC + 001450421), Clinical isolates of Staphylococcus aureus (7133623 LACEN - Central Public Health Laboratory) and Staphylococcus aureus (7133623 HRAN) were used. Therefore, the multifunctional antimicrobial peptide showed efficacy in the in vitro assays performed. In addition, Ts-MAP1 showed activity against tumor cell lines in vitro, further demonstrating its multifunctionality. (002) The synthetic peptide presented here represents alternatives for the development of drugs intended for the control of various infections for human health, as well as for agribusiness, through the control of phytopathogenic and pathogenic bacteria in animals, and therefore, falls under the International Patent Classification Section A - Human Necessities, Subsection A61 - Health, Rescue, Recreation, Medical or Veterinary Science, Hygiene, Subsection A61K 38 / 00 - Medicinal preparations containing peptides; Section C - Chemistry, Subsection C07K - Peptides, Subsection 07K 1 / 00 ​​- General processes for the preparation of peptides. DESCRIPTIVE REPORT (003) The discovery of antibiotics represented a major advance in public health, leading to the treatment and cure of infections and increasing human life expectancy (AOKI; UEDA, 2013). In recent years, interest in a clinically relevant group of bacteria that cause high mortality, called ESKAPE (Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) has intensified. Antimicrobial-resistant bacteria with the ability to form biofilms are being increasingly studied because they represent a major public health problem due to the difficulty of treatment, consequently increasing the mortality rate (JENSSEN, 2006). (004) However, in recent years, the emergence of microorganisms resistant to conventional antibiotics has highlighted the need for the development of new, more efficient drugs (HERNÁNDEZ-APONTE et al., 2011). International projections indicate that if nothing more effective is done than has been done so far, around 2050, 10 million people will die annually worldwide as a direct consequence of bacterial resistance to antimicrobials (FERNANDES et al., 2016). Throughout the Petition 870250098324, dated 10 / 28 / 2025, p. 4 / 18 / 14 evolutionary process microorganisms have also developed adaptive mechanisms or survival strategies. One adaptive mechanism that has potentiated bacterial resistance to drugs is biofilm formation. (005) Biofilms are communities of microbial cells that are surrounded by a matrix of extracellular polymeric substances and associated with biotic or abiotic surfaces (SAUER; RICKARD; DAVIES, 2007). This phenomenon is driven by the survival principle, as a mechanism of adaptation to environmental stress (APARNA; YADAV, 2008). The mechanisms involved in the biofilm formation process can be grouped as adaptive resistance (SCHROEDER; BROOKS; BROOKS, 2017), innate resistance or, of greater concern for modern clinical and agricultural practice; and acquired resistance with horizontal gene transfer (FERNÁNDEZ; HANCOCK, 2012; ANDERSSON; HUGHES, 2011). Many theories have been proposed regarding the dynamics of biofilm formation, but some fundamental steps are agreed upon in all of them, the main ones being: contact, adhesion, microcolonial formation, maturation, and detachment (O'TOOLE; KAPLAN; KOLTER, 2000).These steps are dependent on the influence of the environment and the genetic characteristics of the bacteria (O'TOOLE; KAPLAN; KOLTER, 2000). (006) Frequently, bacteria from the same community of a bacterial biofilm have different minimum inhibitory doses to antibiotics, offering opportunistic pathogens the ability to remain as infectious agents for a long time (ITO et al., 2009). Data related to in vitro assays have indicated that bacterial cells in biofilms (sessile) can become substantially more resistant to the action of antibiotics than those cultivated in plankton (SMITH; DALY; CRAIK, 2011). This is concerning because the formation of microbial biofilms can occur on both natural and artificial surfaces, such as teeth, contact lenses, native and artificial heart valves (causing endocarditis), lungs of patients with cystic fibrosis (causing chronic bronchopneumonia), middle ear in patients with chronic otitis media, joint prostheses, intravenous catheters, and in chronic wounds (HOIBY et al., 2010). (007) Therefore, the need to develop new methods in the treatment of combating or controlling pathogenic microorganisms and tumor cells has resulted in research focused on antimicrobial peptides (AMPs) and their therapeutic potential (HANEY; MANSOUR; HANCOCK, 2017). The mechanism of action of AMPs is different from conventional antibiotics, being considered an attractive alternative for the control of resistant microorganisms (GIULIANI; PIRRI; NICOLETTO, 2007). Furthermore, elucidating the mechanisms of action, selectivity, and specificity is important for the rational design of new generations of AMPs (GIULIANI; PIRRI; NICOLETTO, 2007). Antimicrobial peptides (AMPs) are produced by virtually all living organisms (ZASLOFF, 2002), are constituent molecules of the innate immunity of multicellular organisms, and are expressed in a constitutive or induced manner (PINHEIRO; MACHADO, 2012; ROSCIA et al., 2013), and are considered a first line of defense against pathogen invasions. Petition 870250098324, dated 10 / 28 / 2025, page 5 / 18 3 / 14 (MOOKHERJEE; HANCOCK, 2007; HANCOCK; SCOTT, 2000). (008) AMPs are low molecular weight proteins with a broad antimicrobial spectrum and immunomodulatory activity against infectious bacteria (Gram-positive and Gram-negative), viruses, and fungi (BOPARAI, 2020). Among the most abundant AMPs are those present in venomous animals. AMPs have been investigated as potential drugs, leading to an increase in their applications in medicine and biotechnology (CRAIK et al., 2013). A growth in research with AMPs began in the 1980s due to the discoveries of magainins by Michael Zasloff, insect cecropins by Hans Boman, and human α-defensins by Robert Lehrer (TOSSI; SANDRI, 2002). Since then, databases such as the Antimicrobial Peptide Database (APD, https: / / aps.unmc.edu / home), last updated in July 2022, have isolated and characterized AMPs in the phylum Arthropoda, with 93 of them found in scorpions. (009) Tityus serrulatus is a scorpion typical of the southeastern, central-western, and northeastern regions, also popularly known as the yellow scorpion. It is considered the most dangerous in South America and is responsible for the highest rate of fatal cases involving venomous animals. Its toxin is composed of several substances of medical importance, among which neurotoxins, inorganic salts, enzymes, and antimicrobial peptides stand out (BUCARETCHI, 1995). The venom of scorpions of the genus Tityus constitutes a complex mixture of enzymes, peptides, nucleotides, lipids, mucoproteins, biogenic amines, phospholipases, hyaluronidase, high and low molecular weight proteins, and other biologically active components. These molecules can exhibit toxic activity and / or contribute to the distribution of toxins in prey (ALMEIDA et al., 2012; BATISTA et al., 2007; ZENG et al., 2004). (010) Secondary structure, cationicity, hydrophobicity, and amphipathicity are the key elements that allow the characterization of antimicrobial peptides, and because of this they are considered excellent candidates for the future development of a new class of antimicrobials to combat and / or control the emerging threat of diseases caused by multidrug-resistant bacteria (HANEY; MANSOUR; HANCOCK, 2017; WANG, 2010; ZASLOFF, 2002), HIV-1 and cancer (ZASLOFF, 2002; WANG, 2010). (011) PAMs present several characteristics favorable to the creation of new antibiotics (PUSHPANATHAN; GUNASEKARAN; RAJENDHRAN, 2013) such as: difficulty in the development of microbial resistance, synergism with conventional antibiotics and neutralization of endotoxins (GIULIANI; PIRRI; NICOLETTO, 2007). In addition, the use of PAMs in therapy offers many advantages, since they possess desirable properties, such as potent bioactivity, high specificity and binding affinity with the receptor and relatively low toxicity (CRAIK et al., 2013). Petition 870250098324, dated 10 / 28 / 2025, page 6 / 18 4 / 14 DETAILED DESCRIPTION OF THE INVENTION (012) The present invention relates to the construction of a synthetic peptide Ts-MAPI (SEQ ID NO 1) (NH2-FLKMIPRLIKGLISAFK-COOH) for the control of bacterial infections, anti-inflammatory with controlled cost without exploiting natural resources. (013) The criterion used to obtain the new analog sequence was the point substitution of amino acid residues in the “control” peptide with the intention of increasing the total net charge, adjusting hydrophobicity, and reorganizing the hydrophobic moment. In addition, the helix diagram of the “control” was used as a support to segregate the hydrophilic and hydrophobic residues, which were arranged in an α-helix leading to the new peptides. Studies have shown that these physicochemical properties of peptides are interrelated so that modifications intended to alter one parameter can result in significant changes to one or more of the others. Understanding and controlling these interrelationships may be the key to designing new peptides with greater potency and specificity (YEAMAN; YOUNT, 2003). (014) The control sequence FLGMIPGLIGGLISAFK (TsAP-2) has a net charge of +1 and hydrophobicity of 64%. When the three non-positive amino acids of glycine were replaced by the amino acids lysine and arginine (positively charged), the net charge of the new sequence FLKMIPRLIKGLISAFK (Ts-MAP1) became +4 and the hydrophobicity remained at 64%. The hydrophobic moment increased from 0.594 to 0.749. The physicochemical properties of these peptides were predicted using the HeliQuest server (GAUTIER et al., 2008). The generated sequence was then synthesized and tested in vitro against pathogenic bacteria, healthy cells, and mouse ethrocytes. The three-dimensional models were predicted using the / TASSER software (l-TASSER, https: / / zhanqlab.ccmb.med.umich.edu / l-TASSER / ) (ZHENG, 2021). OBTAINING PEPTIDES (015) The synthetic peptide Ts-MAP1 was synthesized by the company Aminotech using the solid phase methodology, F-moc (9-fluorenylmethyloxycarbonyl) with a purity greater than or equal to 95%. MINIMUM INHIBITORY CONCENTRATION (MIC) (016) MIC assays were performed against strains of A. Bauman nii (003324845 HRAN - Hospital Regional Asa Norte), E. coli clinical isolate (3323742), E. coli (KPC + 001812446), K. pneumoniae (ATCC + 13883), K. pneumoniae (KPC + 001450421), S. aureus clinical isolate (7133623 LACEN - Laboratório Central de Saúde Pública) and S. aureus clinical isolate (7133623 HRAN). The bacteria were plated on Mueller Hinton agar (MHA) plates and incubated at 37 °C overnight. After this period, three isolated colonies of each bacterium were inoculated into 5 mL of Mueller Hinton broth (MHB) and incubated at 200 rpm, 37 °C, overnight. Bactehano growth was Petition 870250098324, dated 10 / 28 / 2025, page 7 / 18 / 14 monitored by spectrophotometer at 600 nm. MIC tests were performed using the 96-well microplate dilution method at a final bacterial concentration of 5x10⁶ CFU.mL⁻¹. Peptides were tested in a concentration range for TsAP2 (2.3 to 73.8 μM) and for Ts-MAP1 (2.0 to 64.8 μM). The antibiotic ciprofloxacin was used as a positive control at the same concentrations, while the bacterial suspension in MHB was used as a negative control. Microplates were incubated at 37 °C for 18 hours and readings were taken on a Multiskan Go microplate reader (Thermo Scientific) at 600 nm after the incubation time. The MIC was determined as the lowest peptide concentration at which no significant bacterial growth occurred. MINIMUM BACTERICIDAL CONCENTRATION (MBC) (017) The evaluation of the minimum bactericidal concentration (MBC) was dependent on the MIC results against bacteria. Three 10 μL replicates were taken from the microplate wells, plated on MHA and incubated at 37°C for 24 h. The MBC was determined as the lowest peptide concentration at which no bacterial growth was detected. All tests were performed in triplicate (WIEDERSTEIN; SIPPL, 2007). MEMBRANE PERMEABILIZATION USING SYTOX GREEN (018) Bacterial membrane permeability was investigated using the Sytox green dye, as described by Mohanran and colleagues, with modifications proposed by (ALMEIDA et al., 2021; MOSMANN, 1983). In the assay, A. baumannii (003324845 HRAN) and S. aureus (7133623 LACEN) were cultured in Mueller Hinton Broth for 18 h at 37°C, prepared at an optical density of 600 nm of 0.5, in 10 mM sodium phosphate buffer, pH 7.0. Next, 280 pL of bacterial suspension were transferred to 96-well black microplates, where 10 pL of Sytox green at 123 pM (30 x MIC) were added and incubated for 10 min at 37°C. Subsequently, 10 pL of TsMAP1, at a concentration 30 times higher than the MIC, were added to each well, and the kinetic assay was performed for 50 min, with readings every 5 min.The assay was performed using fluorescence reading, excitation at 485 nm and emission at 520 nm, in a Varioskan Lux microplate reader (Thermo Scientific: Waltham, MA, USA). Negative control of membrane damage was performed with bacteria incubated with 10 µL of 10 mM sodium phosphate buffer, pH 7.0. Three independent experiments were performed in triplicate. SURFACE CHARGE MEASUREMENTS (ZETA POTENTIAL) (019) Zeta potential studies were performed at 25°C from the average of 15 measurements (100 runs each), in the absence and presence of different peptide concentrations, using disposable DTS 1070 zeta cells with gold electrodes (Malvern, UK), after a 15-minute equilibration time. The data were processed using Malvern's DTS software, after three independent experiments (FELÍCIO et al., 2021). For the assay, A. baumanni (003324845 HRAN) and S. aureus (7133623 LACEN) Petition 870250098324, dated 10 / 28 / 2025, page 8 / 18 / 14 were cultured in Mueller Hinton Broth for 18h at 37°C, prepared at an optical density of 0.5 of 600 nm, in 10 mM sodium phosphate buffer, pH 7.0. The Ts-MAP1 peptide was prepared at 123 μM (30 x MIC). MINIMUM INHIBITORY CONCENTRATION OF BIOFILM (MIBC) (020) Biofilm formation was obtained using Luria-Bertani medium, abbreviated LB medium, in a 96-well round-bottom cell culture microplate, in the presence of TsAP-2 (2.3 to 73.8 μM) and Ts-MAP1 (2.0 to 64.8 μM) peptides at 37°C for 24 hours. The positive growth control contained only bacteria and the negative growth control wells contained only LB broth. After the incubation time, planktonic cells were removed and the microplate wells were washed twice with deionized water. The remaining adherent bacteria were stained with 100 μL of 0.1% (w:v) crystal violet for 20 minutes. The microplates were washed twice with deionized water, air-dried, and solubilized with 100 μL of 60% ethanol. The contents of the microplate were transferred to a new microplate, and the MBIC of the peptides was recorded at 595 nm using a Multiskan Go microplate reader (Thermo Scientific).Three independent experiments were performed for each condition tested. BIOFILM ERADICATION (021) A pre-formed biofilm of A. baumannii (003324845 HRAN) and S. aureus (7133623 LACEN) was obtained as previously reported, with some modifications (SILVA et al., 2023). The microbial culture was grown at 37°C to an optical density of 1.6 (λ = 600 nm) and then diluted to a cell density of 5 x 10⁵ CFU-mL⁻¹. 150 μL aliquots were dispensed into the wells of a 96-well plate, which was incubated for 18 h at 37°C for biofilm formation. After the incubation period, the medium containing planktonic cells was aspirated from the wells and replaced with 100 μL of phosphate-buffered saline (PBS) to remove any non-adherent cells. A double wash with PBS was performed. After washing, each well was filled with PBS supplemented with different serial dilutions of Ts-MAP1 (from 3.8 to 123 μM), and the plate was then incubated for 2 h at 37°C.Following peptide treatment, the wells were washed twice with PBS, as indicated above, and 20 μL of MTT (0.5 mg.mL-1) were dispensed into each well to assess biofilm cell viability. A higher intensity of purple color corresponds to a higher percentage of metabolically active cells and, consequently, greater cell viability. The plate was incubated and protected from light at 37°C for 3 hours, and the reaction was stopped by the addition of the solubilized solution. The absorbance of each well was recorded at 570 nm using a Multiskan Go microplate reader (Thermo Scientific, Waltham, MA, USA), and the percentage of biofilm viability was calculated relative to untreated samples. Petition 870250098324, dated 10 / 28 / 2025, page 9 / 18 / 14 ANTIFUNGAL ACTIVITY (022) Candida albicans strains (ATCC 90029), Candida tropicalis (ATCC 750), Candida krusei (ATCC 6258), Candida parapsilosis (ATCC 22019), Candida glabrata (ATCC 9030), Candida auris CBS 10913 and four Candida auris (2015 / 466, 2015 / 467, 2015 / 468, 2015 / 470) clinical isolates from a Venezuelan outbreak. The strains were stored at -80°C in nutrient broth supplemented with 2% glycerol. Before experimentation, each strain was seeded on Sabouraud Dextrose Agar (SDA) plates and incubated overnight at 37°C. The antifungal activity of the peptide was determined according to a standardized broth microdilution method (Clinical and Laboratory Standards Institute (CLSI - M27-A2) document). Briefly, yeast colonies were resuspended in 5 mL of sterile 0.145 mol.L-1 saline solution and adjusted to a cell density of 1x106 to 5x106 cells per mL-1. The yeast suspension was then diluted to obtain an initial inoculum of 5x102 to 2.5x103 cells.mL -1. The Ts-MAP1 peptide was dissolved in water and prepared to a stock concentration of 2 mg.L-1, followed by serial dilution in RPMI 1640 growth medium buffered with 3-morpholinopropane-1-sulfonic acid (MOPS), in a volume of 30 μL per well, obtaining final concentrations ranging from 64.8 to 1.0 μM in 96-well microplates with a sterile bottom. A volume of 30 μL of standardized yeast suspension was added to each well. The plates were incubated for 48 h at 37°C and monitored at 530 nm. The MIC was defined as the lowest concentration that inhibited 90% of Candida species growth. HEMOLYTIC ASSAY (023) Hemolytic assays were performed with fresh mouse blood, collected and centrifuged to separate the red blood cells, and washed with 0.9% saline solution. The hemolytic effect of the peptides was evaluated by exposing erythrocytes to different serial dilutions of these molecules. Peptide solutions were added to the erythrocyte suspension (1% by volume) at varying concentrations (from 2.3 to 73.8 μM) for TsAP-2. For Ts-MAP1, the concentration was made using 30 x MIC (minimum inhibitory concentration) previously tested (from 3.8 to 123 μM). 0.9% saline solution and Triton X-100 were used as negative and positive controls, respectively. After exposure, hemoglobin release was measured at 415 nm (PARK et al., 2004). All tests were performed in triplicate. This experiment was approved by the Ethics Committee on Animal Use (CEUA) of the Dom Bosco Catholic University (UCDB) under number 014 / 2022.Three independent experiments were conducted for each condition tested. CELL CULTURE AND CELL VIABILITY (024) To evaluate the cytotoxicity of the peptides, a 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay was performed, following a modified protocol derived from (KUMAR; NAGARAJAN; UCHIL, 2018). The tumor cell lines used were NCI-H292 (ATCC CRL-1848), RD (ATCC CCL-136), HeLa (ATCC CCL2), and healthy MRC-5 (ATCC CCL-171), acquired from the Cell Culture Center of Petition 870250098324, dated 10 / 28 / 2025, page 10 / 18 / 14 Adolf Lutz Institute (São Paulo, Brazil). Two murine cell lines, BV2 and Raw 264.7, were also used. The cell lines were maintained under sterile conditions, following the instructions provided. Cultured in DMEM (SIGMA) with 10% fetal bovine serum (SIGMA), the cells were incubated at 37°C in an environment with more than 95% humidity and 5% CO2 in a 75 cm² culture flask (KASVI). When they reached 90% confluence, the cells were detached, centrifuged at 970 g for 5 minutes at room temperature, and then seeded at 2x10⁵ cells per well in a 96-well plate. The Ts-MAP1 concentration was determined using a previously tested 30 x MIC (minimum inhibitory concentration) (from 1.9 to 123 μM) and incubated at 37°C for 24 hours. After discarding the supernatant, a 5 mg.mL-1 MTT solution (SIGMA) diluted in PBS was applied to each well. The covered plate was incubated for 4 hours at 37°C in the dark.Formazan crystals were solubilized using a solution of hydrochloric acid and isopropyl alcohol, and cell viability percentages were determined at 540 nm using a microplate reader (Thermo Scientific Multiskan Britain). The results were calculated relative to untreated control cells. Cell Viability (%) = (Abs Sample / Abs Positive Control) x 100 NITRIC OXIDE (NO) DOSAGE (025) NO production was quantified according to the Griess reaction method (STONE; YANG; QUI, 2005). Peritoneal macrophages (2 x 10⁵ cells.mL) were incubated with DMEM medium for 24 hours for adhesion. After that, the cells were incubated with different concentrations of Ts-MAP1. The assay was performed using 30 x previously tested MIC (minimum inhibitory concentration) (from 1.9 to 123 μM) and LPS (200 ng / well). After 24h, the supernatant was collected and quantified using the Griess reaction method. The supernatant was removed from the plate and 50 μL was incubated with Griess reagent. All tests were performed in triplicate. The reading was obtained at 570 nm (SpectraMax F3, Molecular Devices). The results were compared with a nitrite curve (200 to 1 μM) and expressed in μM.mL. CIRCULAR DICHROISM (CD) SPECTROSCOPY (026) Circular dichroism (CD) analyses were performed on a Jasco J-1100 spectropolarimeter (Jasco Inc., Japan) using a quartz cuvette with a 1 mm optical path. The spectrum from 260 to 185 nm was collected in steps with a resolution of 0.1 nm to 100 nm.s, at 25°C, with an average of 5 cumulative scans for each spectrum. The Ts-MAP1 peptide was prepared in a 120 pM stock solution and incubated under different conditions at a concentration of 30 pM. The secondary structure of the Ts-MAP1 peptide was analyzed in the presence of water, 50% trifluoroethanol (TFE), or 30 mM SDS. The data were converted to molar ellipsity (0), according to the equation: Petition 870250098324, dated 10 / 28 / 2025, page 11 / 18 / 14 [0] = ----10 * C * 1 * nr (027) Where, Θ is the ellipsity measured in milliseconds, C is the peptide concentration (M), l is the cuvette path length, and nr is the number of amino acid residues. The fractional alpha-helix content, hn, was estimated using the equation: 0222 - OC (028) Where, Θί= 2220-53, ΘH� = (250 T - 44000) (1-3 / n), where T is the temperature in Celsius, and n is the number of amino acid residues in the peptide. The values ​​Θί and ΘH� represent, respectively, the limiting values ​​of average ellipsity at 222 nm (Θ222) for a disordered and alpha-helix conformation. SEQUENCE LISTING (029) SEQ ID NO 1= NH2-FLKMIPRLIKGLISAFK-COOH (030) The sequence of the new analog, Ts-MAP1 (NH2-FLKMIPRLIKGLISAFK-COOH), exhibited physicochemical characteristics shared with PAMs, including net charge (+4), hydrophobicity (0.901), hydrophobic residue content (64%), and hydrophobic moment (0.594). Furthermore, the amino acids are distributed through a partially amphipathic α-helix, since the hydrophilic face has a central Ala (hydrophobic) residue surrounded by 4 Lys residues. The hydrophobic face is composed of a combination of Leu and Iso residues (Figure 1A). The three-dimensional model of Ts-MAP1 shows the distribution of 3 Lys and 1 Arg residues at the N and C terminus (Figure 1D). The synthetic peptide Ts-MAP1 was purified by reverse-phase high-performance liquid chromatography (RP-HPLC), with a purity greater than 98%. The mass spectrum of Ts-MAP1 showed a mass of 1,975.55 Da. (031) The antibacterial activity of Ts-MAP1, expressed in MIC and MBC, where the peptide showed MIC against A. baumannii (003324845 HRAN), E. coli (3323742), E. coli (KPC+001812446), K. pneumoniae (ATCC+13883), K. pneumoniae (KPC+001450421), S. aureus (7133623 LACEN) and S. aureus (003730529 HRAN) at concentrations of 4.1, 8.1, 16.2, 4.1, 32.2, 4.1 and 4.1 μM respectively. (032) The evaluation of bacterial membrane damage was performed for the Ts-MAP1 peptide against strains of A. baumannii (003324845 HRAN) and S. aureus (7133623 LACEN), in which the increase in fluorescence of the Sytox green dye suggested that the peptide caused damage to the plasma membrane. For TsAP-2, no fluorescence was observed at the end of 100 min. Ts-MAP1 on the A. baumannii membrane reached 50% permeabilization in 30 min, with a peak of 100% observed at 90 min. For S. Petition 870250098324, dated 10 / 28 / 2025, page 12 / 18 / 14 aureus, Ts-MAP1 showed an increase in fluorescence, reaching 50% from 15 min and 100% at 70 min. These data confirm the occurrence of damage to the bacterial membrane but highlight differences in the interaction of TsAP-2 and Ts-MAP1 with Gram-negative and Gram-positive membranes. (033) In the zeta potential assay, A. baumannii (003324845 HRAN) cells showed a negative charge of -47.54 mV and S. aureus (7133623 LACEN) cells -36.16 mV. After 100 minutes of treatment with 123 μM (30 x MIC), the Ts-MAP1 peptide promoted electroneutralization, bringing the membrane potential closer to neutral, causing disruption. For A. baumannii (003324845 HRAN), the surface charge was -16.91 mV and for S. aureus (7133623 LACEN) -24.07 mV. (034) The minimum inhibitory concentration of biofilm (MICB) of Ts-MAP1 was determined against A. baumannii (003324845) at a concentration of 8.1 μM the peptide inhibited 100% biofilm formation. (035) To evaluate the activity of Ts-MAP1 against pre-formed biofilm of A. baumannii (003324845) and S. aureus (7133623 LACEN). The peptide was tested at different concentrations. Ts-MAP1 showed eradication activity at 30.7 μM, leading to the death of ~100% of the biofilm mass of A. baumannii cells. At concentrations of 15.3 to 123 μM, cell mass viability decreased by nearly 100%. For S. aureus, Ts-MAP1 eradicated the biofilm at all concentrations. (036) The antifungal activity of Ts-MAP1, expressed in MIC, where the peptide showed activity against Candida glabrata species (ATCC 9030) at a concentration of 64.8 μM. (037) Hemolytic activity was performed for the TsAP-2 and Ts-MAP1 peptides against murine erythrocytes, wherein the ability to disrupt murine erythrocytes at different concentrations. For the parent peptide, the hemolytic assay was performed (from 2.0 to 64.8 μM). For Ts-MAP1, the hemolytic assay was performed 30 x MIC (3.8 to 123 μM). TsAP-2 did not show hemolysis at any concentration tested. However, the analogous peptide Ts-MAP1 showed approximately 40% hemolysis at 123 μM and demonstrated an IC50 = 6.0 μM. (038) Thus, through the cell viability test, it was possible to perform treatment with the Ts-MAP1 peptide in the Raw 264.7 and BV-2 cell lines stimulated by LPS. The Ts-MAP1 peptide was not cytotoxic at concentrations equal to or below 7.6 μM against Raw 264.7. In relation to the BV-2 cell line, the peptide did not demonstrate any cytotoxicity against microglia cells. In the NO dosage treatment, the peptide against Raw 264.7 cells showed inhibition in NO production between 3.8 and 61.5 μM for macrophages and in the NO dosage against the murine microglia cell line, the Ts-MAP1 peptide inhibited nitrite production between 0.0158 and 1.0 μM. (039) The Ts-MAP1 peptide showed cytotoxicity against HeLa tumor cells. Petition 870250098324, dated 10 / 28 / 2025, pp. 13 / 18 / 14 and RD at 2 μM. The healthy MRC-5 cell line remained viable, demonstrating low cytotoxicity, and was used as a viability control. (040) The structural characteristics of the TsAP-2 and Ts-MAP1 peptides were obtained through circular dichroism (CD) studies, incubating the peptides in solutions mimicking different environments, as can be seen. In aqueous solution, TsAP-2 showed a random coli structure characterized by a negative band at 195-210 nm and no signal at ~220 nm. The signal pattern changed when TsAP-2 was incubated with 50% trifluoroethanol (TFE) and 30 mM SDS micelles, showing positive bands at approximately 192 nm and negative bands at 208 and 222 nm. The changes in CD signal and the presence of negative bands at 208 and 222 nm indicate a conformational transition indicating α-helix. The analog peptide Ts-MAP1 showed a random coil conformation in an aqueous environment. In TFE and SDS assays, the peptide showed positive bands at approximately 192 nm and negative bands between 208 and 222 nm, indicating the conformational transition to α-helix. LIST OF FIGURES Figure 1 illustrates the rational design strategy of the Ts-MAP1 analog peptide bioinspired by the TsAP-2 parental peptide. Figure 2 presents the results regarding the Ts-MAP1 analog peptide in inhibiting biofilm formation in A. baumannii and S. aureus strains. Figure 3 shows the effect on permeabilization of the analog peptide Ts-MAP1 on the membranes of A. baumannii and S. aureus. Figure 4 presents the results regarding the Ts-MAP1 analog peptide against biofilm eradication of A. baumannii and S. aureus strains. Figure 5 shows the hemolytic effect of the analog peptide Ts-MAP1 against murine erythrocytes. Figure 6 shows the cytotoxic effect of the analog peptide Ts-MAP1 against murine macrophages RAW 264.7. Figure 7 shows the cytotoxic effect of the analog peptide Ts-MAP1 against murine BV-2 microglia. Petition 870250098324, dated 10 / 28 / 2025, page 14 / 18 / 14 Figure 8 shows the inhibition of nitric oxide (NO) production in the RAW cell line. 264.7 for the analogous peptide Ts-MAP1 Figure 9 shows the inhibition of nitric oxide (NO) production in the BV2 cell line for the Ts-MAP1 analog peptide. Figure 10 shows the cytotoxic effect of the Ts-MAP1 analog peptide against the healthy MRC-5 cell line and the tumor cell lines RD, NCI-H292, and HeLa. Figure 11 shows the structural behavior through circular dichroism in response to different mimetic environments. REFERENCES (041) AOKI, W.; UEDA, M. Characterization of Antimicrobial Peptides toward the Development of Novel Antibiotics. Pharmaceuticals, vol. 6, no. 8, p. 1055-1081, Aug. 2013. (042) APARNA, MS; YADAV, S. Biofilms: microbes and disease. Brazilian Journal of Infectious Diseases, v. 12, no. 6, p. 526-530, Dec. 2008. (043) BOPARAI. 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Claims

(001) MULTIFUNCTIONAL EVALUATION OF THE SYNTHETIC PEPTIDE BIOINSPIRED BY TITYUS SERRULATUS TOXIN, characterized by having a synthetic peptide, SEQ ID NO 1 / Ts-MAP1 (NH2-FLKMIPRLIKGLISAFK-COOH) (002) MULTIFUNCTIONAL EVALUATION OF THE SYNTHETIC PEPTIDE BIOINSPIRED BY TITYUS SERRULATUS TOXIN, characterized by having 17 amino acid residues that confer a cationic character of +4 charge and 64% hydrophobicity in its peptide sequence (003) MULTIFUNCTIONAL EVALUATION OF THE SYNTHETIC PEPTIDE BIOINSPIRED BY TITYUS SERRULATUS TOXIN, characterized by having antibacterial activity in the presented peptide against Acinetobacter baumannii (003324845 HRAN), Escherichia coli clinical isolate (3323742), Escherichia coli (KPC + 001450421), Klebsiella pneumoniae (ATCC + 13883), Klebsiella pneumoniae (KPC + 001450421), Staphylococcus aureus clinical isolate (7133623 LACEN) and Staphylococcus aureus clinical isolate (7133623 HRAN).It showed activity against the Candida glabrata strain (ATCC 9030) and HeLa and RD tumor cell lines, and exhibited anti-inflammatory activity.

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